Reviewed by Larry H. Title: Pgbr-22: Steps taken towards Characterization of a protein
Introduction: [[#|Recombinant proteins]] are commonly used in the biological sciences, and aid in research for biomedical processes. One problem many scientists initially had was where to begin with a protein. Questions arose such as what length of protein should be used? Which strain should be chosen? What medium should it be expressed in? Each protein is different, and a general place to start (Start what?) is hard to pin down, but through collective research scientists (Don't say "scientists", it is vague) have come up with a good starting point, and have experimented to provide initial data to other researchers [1] (This last part is very vague. What was the experiment? What initial data? Which researchers?). Typically the starting point in this process (Clarify which process) is expression of a protein, to create enough of it to work with. This is followed by Purification of the protein to remove any extraneous materials or other proteins not specific to the project. For example purification can be done by histones (Histidine?). By coating proteins in a specific chemical (What chemical?), they can be attracted to the histones, and let the rest of the solution filter out. The [[#|last]][[#|step]] is known as expression (purification), in which gel chromatography is used to further purify the protein, and isolate it in a way that it can be compared to a standard ladder, or reference protein (This is done with SDS-PAGE gels, not gel chromatography). During this lab, the goal is to accomplish all three of these steps in hopes of getting a purified comparable sample in the end. It was hypothesized that the protein would be slightly different from the ladder (different in what way?), due to user error, and other possible issues that might come up during the lab.
M&M comments: Pgbr-22 is a plasmid. Be more specific with what materials you use, for example, give concentrations, company names, etc. What kind of gel did you use? Materials & Methods: Expression of the protein was the first step in the lab process. This was achieved by heat-shocking E. coli bacteria, and then having them intake the Pgbr-22 protein selected. The colonies were then grown on an Agar Amp transformation plate. After waiting 8 hours for the bacteria to culture it was transferred to a transformation tube and then put back into the shaker. This process was repeated to achieve a large culture. The solution was put into conical tubes and then centrifuged, and a pellet was formed and kept for the next step. The second step in the process was the purification of the Pgbr-22 protein. One of the conical tubes containing the pellet was thawed, and then had lysozyme mixed in to digest the [[#|cell walls]]. Cyanase was later added to reduce the viscosity of the solution. Two buffers were created and labeled Wash and Elution buffer. A chromatography tube was used along with the buffers to filter the Pgbr-22 protein. The Nanodrop spectrophotometer was then used to measure the concentration of the protein used. Multiple samples were taken during this process and throughout the lab to be used in the final step. The final step was to characterize the protein in question. The six samples collected during the previous two labs were all mixed with a dye, and then heated. A gel electrophoresis machine was prepared, and the samples were injected onto the gel. A ladder was also added for comparison. The gel was run, and rinsed in the shaker overnight. Finally the gel was dried and compared to the ladder online to determine the concentration and purity of the Pgbr-22 protein.
Results:
Figure1. Initial transformation plate with bacteria spread on surface. Picture taken before incubation.
Figure2. Transformation plate after incubation. Colonies present, and ready to be used for next incubation period.
Were these controls? Be more specific about what this picture shows. i.e. incubation period, what types of colonies, what kind of plates
Figure3. Flask containing desired bacterial culture. Notice the color has changed to a light purple indicating that the bacteria has taken up the desired protein and replicated.
The bacteria takes up plasmid, not protein.
Figure4. Conical tube containing bacterial pellet after the solution had been centrifuged and the liquid drained.
Figure5. Elution tube 1, taken after the initial wash in the chromatography column. The solution is mostly a light purple indicating the presence of our protein.
Figure6. Elution tube 2, taken after the second wash through the chromatography column. The color is clear meaning the majority of the protein was filtered during the first wash.
The term "wash" refers to a specific step in the purification process.
Figure7. Nanodrop spectrophotometer readings of the protein at 280nm. The path length was one cm (10mm) and was used later in the beer's law calculation.
Figure8. Final (dried) gel showing the ladder, and following samples 1-6 from partner 1 and 4-6 from partner 2.
Figure9. Comparative chart showing the actual ladder used, and various readings for size of the protein in kilodaltons.
Label your lanes like this: Lane1: blah Lane2: blahblah etc...
Figure10. Beer's law calculations after purification of the protein, and using absorbency reading from Nanodrop, using a path length of 1cm.
Discussion: Overall the lab went very smoothly. During each process the end result was achieved and could be carried over to the next [[#|step]]. However, there were a few possible sources of error that although small could've had an impact on the lab as a whole. Many of the possible errors or contamination hazards that could have occurred would have taken place in the first step, expression. This was one of the most important steps, and the work area needed to be kept sterile as long as possible. To accommodate this, gloves were used at all times, and any time the bacteria was being transferred an open flame was nearby to sterilize the general area. Other precautions such as quickly exposing the pipette tips to the flame helped to reduce contamination. The other two processes, purification and expression, didn't need to be sterilized, but mistakes such as administering proper amounts of solutions could be made. The results were on track with what was expected at the end of the lab, and the protein fell right between 15-25 kilodaltons as can be seen in figure 8. Unfortunately the banding didn't appear as dark as the ladder in comparison so the protein wasn't as purified as had been hoped for. Another concern was that samples 4, 5, 6, and the second sample 6 were all very light compared to the rest of the bands. This was probably due to the samples used. In the previous lab the correct amount of sample (50 microliters) wasn't collected, which caused diluting in the samples after the dye had been added. In order to compensate for this mistake the gel was left to stain longer than normal. This may not have caused a big change, but all of the bands are still visible for comparison. During the purification of the protein, lysozyme was used to digest the cell walls of any of the bacteria remaining in solution. The Cyanase reduced the viscosity of the bacteria by digesting the DNA/RNA in the mixture. The Histadine tags worked by sticking to the nickel that was added during the elution process. Because Nickel is attached to the histadine, it is too large, and gets stuck to the resin instead of filtering through with the protein. The nickel doesn't go through the column since they are attached to large beads. During the purification process the protein was being moved around constantly, going from in the mixture during step one, to being in the solution with DNA/RNA in sample 2. The protein further progressed to the transfer tube during step 3 and was then attached to a histadine. After being washed, initially it was still in the solution, but was then transferred to elutions 1 and 2 for the last samples taken. Mention imadazole The Wash and elution buffers were very similar, however, what truly made the difference between the two was the strength of the solution. The actual wash buffer was much weaker weaker in what way? compared to the elution buffer, which is why the elution was used to strip away the protein. The protein examined in the lab was Pgbr-22, and when looking at the ladder, it can be inferred that the size of the protein was between 15-25 kilodaltons, most likely right around 22-23 kilodaltons. The size of the protein determined in the purification lab was larger than the value found in this experiment, and was likely due to possible error. The purity of the sample in figure 8 is weaker than the ladder as can be seen by the darker banding pattern of the ladder. Although one or two of the samples did have a darker banding patter, the majority of them had very faint lines, and couldn't be seen extremely well. This data in turn says that the overall purity of the protein is relatively weak, and below 50% as compared with the ladder.
Talk about elution 1 vs. elution 2. What do you expect the banding pattern for elution 2 to be?
Conclusions:
During lab, the protein Pgbr-22 was experimented on and taken through the processes of getting to a state in which it could be characterized. The protein was expressed over a three day period, involving bacterial culturing in two different groups. The following weeks then consisted of a purification process in which the protein was filtered and multiple samples were taken to be tested on later. The last step in the process was the characterization of the protein by gel electrophoresis. When all steps had been completed, it was found that the protein in question was slightly smaller than the calculated value found during the protein purification lab. Other data collected included the size of the protein we tested. Although this value is already known, it is important that we compare what we found to the actual value to compensate for any mistakes or errors made during all parts of the lab. The skills that were used to complete this lab can later be used in the future to characterize other proteins. The process would probably take less time, and now knowing all the techniques less errors would be made. Although wet-lab is a good way to learn, it is time consuming and expensive depending on the protein tested and materials used, so for further testing it would be a good idea to know for a fact which proteins need to be tested and what we are trying to characterize them for. This is where virtual screening comes in, and this along with the PyMOL program can assist in analyzing a protein. Why do we need purified protein?
References: 1. Structural Genomics Consortium. China Structural Genomics Consortium. Northeast Structural Genomics Consortium. Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schütz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus KC. Protein production and purification.Nat Methods.2008;25:135
Title:
Pgbr-22: Steps taken towards Characterization of a protein
Introduction:
[[#|Recombinant proteins]] are commonly used in the biological sciences, and aid in research for biomedical processes. One problem many scientists initially had was where to begin with a protein. Questions arose such as what length of protein should be used? Which strain should be chosen? What medium should it be expressed in? Each protein is different, and a general place to start (Start what?) is hard to pin down, but through collective research scientists (Don't say "scientists", it is vague) have come up with a good starting point, and have experimented to provide initial data to other researchers [1] (This last part is very vague. What was the experiment? What initial data? Which researchers?). Typically the starting point in this process (Clarify which process) is expression of a protein, to create enough of it to work with. This is followed by Purification of the protein to remove any extraneous materials or other proteins not specific to the project. For example purification can be done by histones (Histidine?). By coating proteins in a specific chemical (What chemical?), they can be attracted to the histones, and let the rest of the solution filter out. The [[#|last]] [[#|step]] is known as expression (purification), in which gel chromatography is used to further purify the protein, and isolate it in a way that it can be compared to a standard ladder, or reference protein (This is done with SDS-PAGE gels, not gel chromatography). During this lab, the goal is to accomplish all three of these steps in hopes of getting a purified comparable sample in the end. It was hypothesized that the protein would be slightly different from the ladder (different in what way?), due to user error, and other possible issues that might come up during the lab.
M&M comments:
Pgbr-22 is a plasmid. Be more specific with what materials you use, for example, give concentrations, company names, etc. What kind of gel did you use?
Materials & Methods:
Expression of the protein was the first step in the lab process. This was achieved by heat-shocking E. coli bacteria, and then having them intake the Pgbr-22 protein selected. The colonies were then grown on an Agar Amp transformation plate. After waiting 8 hours for the bacteria to culture it was transferred to a transformation tube and then put back into the shaker. This process was repeated to achieve a large culture. The solution was put into conical tubes and then centrifuged, and a pellet was formed and kept for the next step. The second step in the process was the purification of the Pgbr-22 protein. One of the conical tubes containing the pellet was thawed, and then had lysozyme mixed in to digest the [[#|cell walls]]. Cyanase was later added to reduce the viscosity of the solution. Two buffers were created and labeled Wash and Elution buffer. A chromatography tube was used along with the buffers to filter the Pgbr-22 protein. The Nanodrop spectrophotometer was then used to measure the concentration of the protein used. Multiple samples were taken during this process and throughout the lab to be used in the final step. The final step was to characterize the protein in question. The six samples collected during the previous two labs were all mixed with a dye, and then heated. A gel electrophoresis machine was prepared, and the samples were injected onto the gel. A ladder was also added for comparison. The gel was run, and rinsed in the shaker overnight. Finally the gel was dried and compared to the ladder online to determine the concentration and purity of the Pgbr-22 protein.
Results:
Lane1: blah
Lane2: blahblah
etc...
Discussion:
Overall the lab went very smoothly. During each process the end result was achieved and could be carried over to the next [[#|step]]. However, there were a few possible sources of error that although small could've had an impact on the lab as a whole. Many of the possible errors or contamination hazards that could have occurred would have taken place in the first step, expression. This was one of the most important steps, and the work area needed to be kept sterile as long as possible. To accommodate this, gloves were used at all times, and any time the bacteria was being transferred an open flame was nearby to sterilize the general area. Other precautions such as quickly exposing the pipette tips to the flame helped to reduce contamination. The other two processes, purification and expression, didn't need to be sterilized, but mistakes such as administering proper amounts of solutions could be made. The results were on track with what was expected at the end of the lab, and the protein fell right between 15-25 kilodaltons as can be seen in figure 8. Unfortunately the banding didn't appear as dark as the ladder in comparison so the protein wasn't as purified as had been hoped for. Another concern was that samples 4, 5, 6, and the second sample 6 were all very light compared to the rest of the bands. This was probably due to the samples used. In the previous lab the correct amount of sample (50 microliters) wasn't collected, which caused diluting in the samples after the dye had been added. In order to compensate for this mistake the gel was left to stain longer than normal. This may not have caused a big change, but all of the bands are still visible for comparison. During the purification of the protein, lysozyme was used to digest the cell walls of any of the bacteria remaining in solution. The Cyanase reduced the viscosity of the bacteria by digesting the DNA/RNA in the mixture. The Histadine tags worked by sticking to the nickel that was added during the elution process. Because Nickel is attached to the histadine, it is too large, and gets stuck to the resin instead of filtering through with the protein. The nickel doesn't go through the column since they are attached to large beads. During the purification process the protein was being moved around constantly, going from in the mixture during step one, to being in the solution with DNA/RNA in sample 2. The protein further progressed to the transfer tube during step 3 and was then attached to a histadine. After being washed, initially it was still in the solution, but was then transferred to elutions 1 and 2 for the last samples taken. Mention imadazole The Wash and elution buffers were very similar, however, what truly made the difference between the two was the strength of the solution. The actual wash buffer was much weaker weaker in what way? compared to the elution buffer, which is why the elution was used to strip away the protein. The protein examined in the lab was Pgbr-22, and when looking at the ladder, it can be inferred that the size of the protein was between 15-25 kilodaltons, most likely right around 22-23 kilodaltons. The size of the protein determined in the purification lab was larger than the value found in this experiment, and was likely due to possible error. The purity of the sample in figure 8 is weaker than the ladder as can be seen by the darker banding pattern of the ladder. Although one or two of the samples did have a darker banding patter, the majority of them had very faint lines, and couldn't be seen extremely well. This data in turn says that the overall purity of the protein is relatively weak, and below 50% as compared with the ladder.
Talk about elution 1 vs. elution 2. What do you expect the banding pattern for elution 2 to be?
Conclusions:
During lab, the protein Pgbr-22 was experimented on and taken through the processes of getting to a state in which it could be characterized. The protein was expressed over a three day period, involving bacterial culturing in two different groups. The following weeks then consisted of a purification process in which the protein was filtered and multiple samples were taken to be tested on later. The last step in the process was the characterization of the protein by gel electrophoresis. When all steps had been completed, it was found that the protein in question was slightly smaller than the calculated value found during the protein purification lab. Other data collected included the size of the protein we tested. Although this value is already known, it is important that we compare what we found to the actual value to compensate for any mistakes or errors made during all parts of the lab. The skills that were used to complete this lab can later be used in the future to characterize other proteins. The process would probably take less time, and now knowing all the techniques less errors would be made. Although wet-lab is a good way to learn, it is time consuming and expensive depending on the protein tested and materials used, so for further testing it would be a good idea to know for a fact which proteins need to be tested and what we are trying to characterize them for. This is where virtual screening comes in, and this along with the PyMOL program can assist in analyzing a protein. Why do we need purified protein?
References:
1.
Structural Genomics Consortium. China Structural Genomics Consortium. Northeast Structural Genomics Consortium. Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schütz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus KC. Protein production and purification. Nat Methods. 2008;25:135
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178102/
2.
European Molecular Biology Laboratory. Protein Expression and Purification Core Facility: Protein Purification. (accessed April 15, 2013)
http://www.embl.de/pepcore/pepcore_services/protein_purification/purification/index.html